Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase which adds deoxyribonucleotide monophosphate to DNA in the absence of a template. TdT is a normal constituent of a subpopulation of immature lymphocytes in the hematopoietic system. TdT is normally expressed in approximately 65% of thymocytes and 1-5% of bone marrow cells yet is virtually absent in circulating lymphocytes. Bollum, J. Biol. Chem., 235:2399 (1960), Coleman, et al, Proc. Natl. Acad. Sci., U.S.A., 71:4404 (1974). As such, TdT is regarded as a biochemical marker for certain immature lymphocytes.
Elevated TdT levels have been reported in the circulation of severely malnourished children. Chandra, et al, Acta. Pediatr. Scand., 68:841 (1979). The rise of TdT levels in these individuals correlates with an increase in levels of immature (null) lymphocytes suggesting that severe nutritional deprivation might retard normal lymphocyte maturation.
Elevated TdT levels have also been described in blood and bone marrow cells of the majority of patients with acute lymphoblastic leukemia. McCaffrey, et al, Proc. Natl. Acad. Sci., U.S.A., 70:521 (1973); Coleman, et al., Cancer Research, 36:120, (1976). In this disease the blast cells have many of the morphological, histochemical, and surface membrane characteristics of immature lymphocytes. Brandt, et al, Cancer, 42:817 (1978); Cressells, et all, Lancet, 2:1307, (1977); Diekel, et al., J. Clin. Invest., 67:725 (1981). In contrast, blasts from patients with acute myelogenous leukemia have only sporadically been reported to contain detectable levels of TdT. Gordon, et al., Blood, 52:1079, (1978); Srivastaya, et al., Cancer Research, 36:3847 (1976).
Conventional techniques for measuring TdT enzymatic activity rely upon a radiopolymerization procedure referred to as a terminal deoxynecleotidyl transferase DNA-polymerization assay. Such TdT DNA-polymerization assays measure the enzymatic activity of TdT, i.e., the ability of TdT to add radiolabeled nucleotide triphosphates to a DNA template, and such assays are limited to measuring TdT enzymatic activity in lymphocyte extracts only.
In addition, it has been reported that TdT enzyme activity is substantially reduced if proper extraction procedures are not employed. Coleman, Archives of Biochemistry and Biophysics, 182, 525-532 (1977) has disclosed that a high salt concentration was mandatory for complete enzyme extraction. Coleman further noted that a buffer containing Tris (pH 7.7), EDTA, 1-mercaptoethanol and 0.5% Triton X-100 was an unsatisfactory extract to preserve TdT enzymatic activity.
It is an object of the present invention to provide a method for the quantitive determination of TdT. In addition, it is an object of the present invention to provide a method for quantitively determining TdT in human blood samples as well as samples obtained from bone marrow extracts and lymphocyte extracts. It is a further object of the present invention to provide a method for extracting TdT from a blood sample wherein antigenic activity of TdT is maintained.